This invention relates to a very simple and convenient immunological detecting method for detecting a component of a biological system, above all for immunological diagnosis, and to a device for immunological detection. More specifically, this invention relates to a method and a device for detecting an antigen or an antibody protein on the basis of an antigen-antibody reaction on a reflecting substrate.
Immunological diagnosis has been performed by utilizing an antigen-antibody reaction which is a very specific biochemical reaction. Radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA) and latex agglutination settling analysis (LSA), for example, are known and used in practice as specific methods of immunological diagnosis. These methods still have technical problems to be solved. RIA has very high detection sensitivity, but requires special facilities for handling radioactive elements. EIA requires a long period of time (usually several hours to one day) for the completion of detection. FIA does not have sufficient detection sensitivity. LSA cannot avoid a non-specific agglutination reaction, and has low reliability in detecting trace components.
On the other hand, an ellipsometric method was proposed in which an increase in the thickness of a protein layer which occurs with the progress of an antigen-antibody reaction on a solid substrate is detected by using ellptic polarized light (British Pat. No. 1,479,661). This method also requires an expensive device and much expertise is required for measuring the protein film thickness. There has also been proposed a method of detecting an antigen-antibody reaction simply with the unaided eye without using such an expensive device. For example, there is a method which comprises adsorbing and fixing an antibody (or an antigen) on and to the surface of gold particles deposited on a solid substrate, and visually observing changes in the color of the reflected light which occur as a result of an increase in the thickness of a layer of an immobilized antibody (or antigen) by an antigen-antibody reaction (U.S. Pat. No. 3,979,184). According to this method, the color of the complex of gold and the protein film on the solid substrate certainly changes with the antigen/antibody reaction. Since, however, the change is only slight from brown to dark brown and very obscure, the evaluation of the antigen-antibody reaction may possibly depend greatly upon the expertise of the testing personnel.
When an antigen or antibody is fixed to a dielectric layer formed on a highly light reflecting substrate such as a metallic chromium or tantalum substrate and an antigen-antibody reaction is carried out on its surface as shown, for example, by Langmuir and Blodgett, Physical Review, vol. 51, pages 964-978 (1937) or Vroman, Thromb. Diath. Haemorrhag., vol. 10, 455-493 (1964), the difference in refractive index between antigen or antibody and the air is very small and the reflectance on the surface of the antigen or antibody is as low as 5% at an incidence angle of 0.degree. to 60.degree.. On the other hand, the proportion of light reflected from the metallic substrate and coming back to the surface of the protein is higher than 50%. Accordingly, it is difficult to detect the interference color on the surface of the device. To discriminate this interference color with good efficiency, it is necessary to adjust the angle of reflection of light on the surface of the device to at least 60.degree. to 70.degree., and it is difficult to detect with the unaided eye. Another proposal for solving this problem (U.S. Pat. No. 4,558,012) states that light interference occurs efficiently by providing two types of dielectric layers on a non-metallic substrate which does not so much reflect light, and making the amount of light reflected from the surface of the substrate nearly equal to that of light reflected from the surface of the dielectric layers. However, substrates meeting such conditions are limited to those which are colored or have high light transmitting property, and those having a high reflectance cannot be used. The colored substrates affect the interference color on the surface of the device and make the detection difficult. With the substrates having a high light transmittance, the color of the device becomes dark and light interference which gives a brilliant visible light color does not easily occur. If a substrate having a relatively high surface reflectance of 60 to 90% is used, it is essential to provide a plurality of dielectric layers having different refractive indices, and the process of building the device becomes complex.